The isolated, perfused rat liver will be used to investigate the mechanism of protein degradation and its regulation by hormonal and nonhormonal agents. Studies completed thus far indicate that when livers from normal, fed rats are cyclically perfused with an unsupplemented medium, large quantities of intracellular protein are taken up and degraded within lysosomes. Although this process is similar to glucagon-induced autophagy, it occurs in the absence of an elevation of cyclic AMP. The rate of processing of intracellular protein by lysosomes will be assessed from the regression of lysosomal contents following the suppression of protein sequestration by insulin and/or amino acids. The results then will be compared with rates of proteolysis in the intact liver measured by valine release. Regression will be assessed both volumetrically (stereologically) and chemically, the latter in highly purified lysosomal fractions isolated after iron-loading. Since autophagy is a cellular response which can be induced both by cyclic AMP and amino acid or insulin lack, we propose to examine the mechanism of regulation in detail and, in particular, to determine whether the cyclic AMP-mediated induction is the consequence of its known action on amino acid utilization or, alternatively, the result of phosphorylation of protein(s) attached to membranes of the lysosomal-vacuolar system. In the latter approach, we plan to take advantage of the density-increasing effect of glycogen and to perfuse neonatal rat livers where free glycogen is the only element taken up. This experimental design should make it possible, after brief exposure to glucagon or cyclic AMP, to isolate autophagosomes which are essentially free of membranes from sequestered organelles.